Encapsulated dye coated noble metal nanoparticles with increased surface enhanced raman scattering properties as contrast agents

ABSTRACT

The present disclosure provides semiconductor-metal composite nanoparticles with optical properties that are superior to those of pure materials for use as contrast agents. The composites include noble metal nanoparticles having a layer of linker molecules being bound to the surface of the noble metal nanoparticle and a layer of dye molecules bound to the layer of linker molecules. The dye molecules are selected such that they form an ordered structure that exhibits a collective absorption band shift, compared to the individual dye molecule, when bound to the noble metal nanoparticle. This structure is encapsulated in a stabilizing coating layer forming a multi-shell structure with properties suitable for biosensing and other detection applications which exhibit enhanced Raman scattering compared to nanoparticles having dye molecules bound thereto not in the ordered structure.

FIELD

The present invention relates to a method for dye coated noble metalnanoparticles with increased surface enhanced Raman scattering (SERS)properties as contrast agents and the use of these as contrast agentswhen encapsulated with a lipid bilayer and having target binding agentsbound to the outer surface of the lipid layer.

BACKGROUND

Contrast agents are used to assist in the identification of molecularand supramolecular targets or regions with distinctive local stateproperties. Non-limiting examples of targeted species are proteins,polysaccharides, polynucleic acids and other analytes, or environmentalproperties. Non-limiting examples of state properties include distinctpH, temperature, and solvent quality. The targeted molecule or state maybe difficult to observe in the background of other material, and thecontrast agent makes it easier to identify it. One example is aparticular type of cell surface protein in the presence of other typesof cell surface proteins. Often such proteins are the markers of ahealth condition, and their identification can assist in diagnosis,determination of treatment or monitoring the progress of a healthcondition. Contrast agents can be used to detect other species, such aschemical threats or biological and chemical warfare agents.

Optical contrast agents, which function using light, are attractivebecause the technology to employ them can be relatively simple, forexample a light microscope or spectrometer coupled to a sampling device.Brighter optical contrast agents can be easier to detect than lessbright agents. Methods to create brighter particles are thereforevaluable. It is useful to have contrast agents that can be easilymultiplexed, meaning that multiple kinds of contrast agents can be usedon the same sample to detect simultaneously the presence and number ofmultiple targets. For example, the type and number of more than one typeof protein might be detected on the surface of a single cell, whichcould improve in identifying the state of the cell and its relevance tothe health of the individual. Optical contrast agents that can bemultiplexed, which means optical contrast agents that can be used in thepresence of other different contrast agents, are therefore valuable.

Plasmonic nanomaterials, and their utility in surface enhanced chemicaland biological sensors, have garnered immense interest in the past twodecades due to their size-dependent optical properties and potential astarget specific contrast agents. Surface-enhanced Raman spectroscopy(SERS) is the most widely studied and offers the possibility of singlemolecule detection.

Raman scattering nanoparticles are potentially useful as opticalcontrast agents because they exhibit sharper optical signatures than,for example, most fluorescent contrast agents. This means that they canin principle be highly multiplexed. Raman scattering by isolatedmolecules is, however, weak, and therefore methods to create brighter ormore strongly scattering Raman contrast agents would be useful.

Raman-scattering nanoparticles have great promise as sensitive detectionlabels. However, due to complex design criteria such as bindingspecificity, robust colloidal stability in biological environments, andoptical sensitivity, few commercially viable sensor systems have beengenerated as a result of this widespread research.

Active plasmonics, defined as plasmonic structures coupled to materialsthat can interact with the plasmon, has generated significant interest,especially in the past few years, as a next generation platform toaddress the need for brighter SERS signals. j-aggregates, Rhodamine 6G,cytochrome c, porphyrin derivatives, and host-guest charge transfercomplexes have all been successfully coupled to plasmonic nanostructuresto elicit a much brighter surface-enhanced resonance Raman (SERRS)signal. Even greater enhancement has been achieved when a wavelengthmatching approach is employed to couple the molecular and plasmonicresonances together with the excitation field. Appropriate coordinationof multiple resonating entities offers a powerful tool for brighterSERS-based sensing platforms. However, while these other approaches havedemonstrated some improvement to the optical brightness, incorporationinto a robust and stable detection label modality has not beenaccomplished.

For practical use of nanoparticles as a detection modality, stability invarious solutions and shelf life are of paramount importance. While inwater, these particles may exhibit desirable properties, but when placedin biological media or buffer solution, they tend to aggregate veryquickly and optical properties are greatly diminished as a result.Often, smaller particles are employed during complicated surfacechemistry, as they are more resistant to aggregation due toelectrostatics, i.e. when charged species or proteins are introducedinto solution and around the particle. However, smaller particles havemuch weaker plasmon resonances, so there is an effective trade-offbetween particle stability and effective optical brightness to make thecomposite particles useful as contrast agents.

Several ways to increase the long term stability of larger SERS-activenanoparticles have been proposed, the most popular being theco-adsorption of various PEG chain lengths to the surface of theparticle along with the Raman dye. Recently, encapsulation of the Ramandye within a stabilizing coating layer that surrounds particles has beenemployed for relatively large particles of 60 nm in diameter. Thesestabilizing layers can consist of inorganic oxides such as silicon ortitanium, or organic self-assembled structures such as lipid vesicles.These composite particles have shown great promise as shelf-stable,biologically compatible biosensors, as the stabilizing coating preventsparticle aggregation in biological media while offering a versatileplatform for targeting functionality and other surface chemistry.

SUMMARY

The present disclosure provides an exitonic semiconductor noble metalcomposite nanoparticle for enhanced Raman scattering, comprising:

a noble metal nanoparticle having a surface;

a layer of linker agents being bound to the surface of the noble metalnanoparticle;

a layer of dye molecules having an absorption band and being bound tothe layer of linker agents, said dye molecules being selected such thatthey form an ordered structure that exhibits a collective absorptionband shift when bound to the noble metal nanoparticle; and

a stabilizing coatinglayer encapsulating the noble metal nanoparticlewith the linker layer and the ordered structure of dye molecules toproduce an encapsulated composite nanoparticle;

wherein said encapsulated composite nanoparticle exhibits enhanced Ramanscattering compared to nanoparticles having dye molecules bound theretonot in said ordered structure.

A further understanding of the functional and advantageous aspects ofthe invention can be realized by reference to the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described, by way ofexample only, with reference to the drawings, in which:

Scheme 1 shows the synthesis of multi-shell 3-aggregate plasmonicnanoparticles. Dye and linkers are conjugated in one step, andsubsequently lipid encapsulated with DEC221 lipid formulation. Particlesare then washed via centrifugation before analysis.

FIG. 1 shows the molecular structure of J-Aggregate forming dye,3,3′-disulfopropyl-5,5′-dichloro-9-ethyl-thiacarbocyanine sodium salt(TC) used to illustrate the enhanced SERS signal according to thepresent invention.

FIG. 1a : Top—Modeled dielectric constant of the J-aggregate layer.Bottom—Field as a function of wavelength for 40 nm diameter particle,0.5 nm linker and 1 nm J-aggregate using FDTD method. Field is plottedat r=21.5, 23.5 and 25.5 nm, respectively.

FIG. 2 shows the electric field intensity as a function of distance fromthe center of the particle. Left side of each plot with 0.5 nm(Thiocholine) TMAT linker and right side is naked (no J-aggregate) forreference.

FIG. 3: a) Naked particle, b,c) with TMAT,(trimethyl-(11-mercaptoundecyl) ammonium chloride) TMA linker layersrespectively coated in TC J-aggregate. Irradiated at 407 nm. d-f)Irradiated at 514 nm. The electric field is strongly confined within theJ-aggregate layer at the 407 nm. The electric field is measured in(V/m)², and the distance from the centre of the particle in nm.

FIG. 4: Top—UV-Vis spectra of J-aggregate and TC. Inset: Molecularstructure of dye. Bottom—UV-Vis spectra of J-aggregate AgNPs with TMATand TMA spacer layers. Inset: Molecular structures of cationic linkers.

FIG. 5 shows Surface-Enhanced (resonance) Raman Spectra of theJ-aggregate functionalized AgNPs. No significant background generated bythe lipid bilayer.

FIG. 6 show typical negative-stain TEM images. Top—Linker/J-aggregatefunctionalized AgNPs, bottom—functionalized particles after lipidencapsulation. Lipid bilayer appears larger than expected due to partialfusing with grid upon sample drying. Scale bar is 20 nm.

FIG. 7 is a UV-Vis plot of the lipid-encapsulated nanoparticles,comparing the spectra 0 days and 21 days after synthesis. Thisdemonstrates the temporal stability of the particles and how the dyeretains the supramolecular structure over a realistic period of time onthe shelf.

DETAILED DESCRIPTION

Generally speaking, the embodiments described herein are directed tolipid encapsulated dye coated noble metal composite nanoparticles, inwhich the dye forms an ordered structure, which exhibit increased SERSsignals. As required, embodiments of the present invention are disclosedherein. However, the disclosed embodiments are merely exemplary, and itshould be understood that the invention may be embodied in many variousand alternative forms.

The figures are not to scale and some features may be exaggerated orminimized to show details of particular elements while related elementsmay have been eliminated to prevent obscuring novel aspects. Therefore,specific method, structural and functional details disclosed herein arenot to be interpreted as limiting but merely as a basis for the claimsand as a representative basis for teaching one skilled in the art tovariously employ the present invention.

Definitions

As used herein, the terms “about”, and “approximately” when used inconjunction with ranges of concentrations, temperatures or otherphysical or chemical properties or characteristics is meant to coverslight variations that may exist in the upper and lower limits of theranges of properties/characteristics.

As used herein, the terms “comprises”, “comprising”, “includes” and“including” are to be construed as being inclusive and open ended, andnot exclusive. Specifically, when used in this specification includingclaims, the terms “comprises”, “comprising”, “includes” and “including”and variations thereof mean the specified features, steps or componentsare included. These terms are not to be interpreted to exclude thepresence of other features, steps or components.

As used herein, the word “linker” or phrase “linker molecule” refers toa species that joins a molecular or molecular aggregate shell to themetal nanoparticle.

As used herein, the word “bound” means joined joined by covalent ornon-covalent bonding, and therefore includes, but is not limited to,bonds where electrons are shared or electrostatic and vander Waalsinteractions, where electrons may not be shared.

As used herein, the phrase “self-assembly” or “supramolecularself-organization” refers to a type of process in which a disorderedsystem of pre-existing components forms an organized structure orpattern as a consequence of specific, local interactions among thecomponents themselves, without external direction. When the constitutivecomponents are molecules, the process is termed supramolecularself-organization or molecular self-assembly.

As used herein, the phrase “J-aggregates” refers to any type of dye withan absorption band that shifts to a longer wavelength (i.e. bathochromicshift) of increasing sharpness (i.e. higher absorption coefficient) whenthe individual dye molecules aggregate to form an ordered structure.Aggregation may be induced from the influence of solvent, additive orconcentration as a result of supramolecular self-organization orself-assembly.

As used herein, the phrase “H-aggregates” refers to_any type of dye withan absorption band that shifts to a shorter wavelength (i.e. ahypsochromic shift) upon aggregation. As noted above, aggregation isinduced from influence of solvent, additive or concentration as a resultof suprarmolecular self-organization.

The present invention is based on the discovery that multi-shellnanostructures comprised of noble metal nanoparticles and dyes that,when bound to the surface of the metal nanoparticle (or bound to alinker layer bound directly to the surface of the metal nanoparticle)form an ordered structure that exhibits a collective absorption bandshift relative to the absorption band of the individual dye molecules.These multilayer structures exhibit novel nonlinear optical propertiesthat can be exploited as a uniquely stable and optically bright sensingplatform. It is possible to employ a wavelength matching approach toimprove SERS response of the metal nanoparticle when used in conjunctionwith this ordered dye structure.

Particularly, the inventors have shown that the electric field can bestrongly confined within the dye ordered structure (monolayer) whenirradiated at the appropriate wavelength as a result of the ENZphenomenon. We experimentally validated the SERS signal as a result offield intensity by varying the effective distance between the resonatingentities. The effect of distance with respect to the particle isdemonstrated experimentally at both resonant and non-resonantwavelengths.

EXAMPLE

The basis of the present invention will be presented herebelow showingthe modeling of the enhanced effect using an exemplary dye, namely a dyewhich forms J-aggregates. J-aggregates are one of the most well-studiedresonant excitonic species available. Under sufficiently highconcentration, or otherwise suitable conditions such as an appropriatelycharged surface, particular cyanine dye molecules will self-assembleinto J-aggregates. J-aggregates generate a collective absorptionresponse that is red shifted from the respective monomeric band, whichgenerates a collective exciton within the aggregate. When these dyes areadsorbed onto noble metal nanoparticles in the j-aggregate assembly, thecollective exciton can couple to the surface plasmon, and interfere bothdestructively and constructively depending on their relative energylevel positions. Ultrafast transient state absorption statespectroscopies as well as theoretical quantum mechanical treatment haveverified the presence of both weak- and strong-type coupling statesbetween the exciton and plasmon. The resonances of these molecules withvarious plasmonic nanostructures have been demonstrated previously, andhave shown that predictable spectral overlap and energetic coupling ispossible. The orientation and adsorption kinetics have also recentlybeen studied in detail, with both thiol-metal bonded andelectrostatic-type adsorption.

The epsilon-near-zero (ENZ) effect has recently garnered interest in thefield of metamaterials, as a way to tailor various sub-wavelengthoptical properties. This effect has been predicted to give very largeenhancement factors for SERS generating nanoparticles. In thisdisclosure the inventors present the first use of ENZ J-aggregatematerials used in coordination with metal nanoparticles to prepare aSERS sensory platform.

Here, the inventors utilize a dye that is traditionally not a good Ramanreporter, but has other unique and useful optical and electricalproperties we exploited to generate a large increase its Ramanscattering.

The dye used in the preferred embodiment of the invention is3,3′-disulfopropyl-5,5′-dichloro-9-ethyl-thiacarbocyanine sodium salt(TC). FIG. 1 shows the molecular structure of J-Aggregate forming dye,3,3′-disulfopropyl-5,5′-dichloro-9-ethyl-thiacarbocyanine sodium salt(TC). However, under suitable conditions, most commonly highconcentration, presence of a multivalent cationic salt, or a cationicsurface, will precipitate self-assembly of the dye into a J-aggregate.What is special about a J-aggregate is it has its own absorption bandwhich is generated by the excitation of a collectively-shared excitonbetween u-stacked rings in aggregate. This absorption is very sharp andnarrow.

It has been shown previously that if the dye can be made toself-assemble into a J-aggregate on a plasmonic nanoparticle, theexciton from the J-aggregate can couple to the plasmon of the noblemetal nanoparticle, given they are sufficiently close enough to oneanother, in what is known as a Fano resonance.

Experimental Methods Materials

Acetylthiocholine, silver nitrate, sodium citrate (99.0%) and hydrogentetracholoroaurate were purchased from Sigma-Aldrich Co. (Canada).Uranyl acetate dehydrate was purchased from Ted Pella, Inc. (USA).N,N,N-trimethyl-(11-mercaptoundecyl) ammonium chloride (TMA) waspurchased from ProChimia Surfaces sp z 0.0. (Poland). TC,3,3′-disulfopropyl-5,5′-dichloro-9-ethyl-thiacarbocyanine sodium salt(TC) was ordered from Hayashibara Biochemical Laboratories, Inc.(Japan). Dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (ESM),and ovine cholesterol (Choi) were received from Avanti Polar Lipids(USA). All chemicals were used as received. Water was purified with aMillipore Milli-Q water system to 18.2 MΩ·cm. All glassware was piranhacleaned.

Synthesis of Silver Nanoparticles (NPs)

The synthesis of the silver nanoparticles (AgNPs) followed the reportedprocedure by Meisel and Lee (Lee, P. C.; Meisel, D. J. Phys. Chem. 1982,86, 3391-3395) Briefly, 6 mg of AgNO₃ was dissolved in 33.3 mL of H₂Oand brought to boil at which point 666 μL of 1% sodium citrate wasadded. The solution was left to reflux for one hour before removing fromheat. The final product was a murky greenish yellow. These were diluted1:3 in water and stored at 4° C. until use.

Thiocholine (TMAT) Synthesis

TMAT was synthesized by simple acid hydrolysis of commercially availableacetylthiocholine in a manner similar to Peng et al. (Peng, L; Zhang,G.; Zhang, D.; Xiang, J.; Zhao, R.; Wang, Y.; Zhu, D. Org. Lett. 2009,11, 4014-7) 500 mg of acetylthiocholine was dissolved in 15 mL ofabsolute ethanol and 4 mL of 37% HCl. While stirring, the solution wasrefluxed at 100° C. for seven hours, after which it was allowed to coolfor 30 min. Excess solvent was removed byrotary evaporation underreduced pressure. Recrystaliization of thiocholine was carried out in aH₂O/isopropanol/ether (0.5 mL/5 mL/25 mL) solvent system. The solutionwas subsequently chilled in an ice bath for 20 min, recovered byfiltration and washed with 25 mL of ether, then allowed to dry. Theresultant white product, herein referred to as TMAT, was dried in adesiccator overnight and stored under argon at −20° C. The product'sstructure was confirmed by NMR (see supporting data) with purity ofapproximately 90%. ¹H NMR (400 MHz, D₂O) δ3.76 (t, J=8 Hz, 2H) 3.23 (m,11H).

Preparation of Ag/Linker/TC Nanoparticles

While stirring, 62.5 μL of 1 mM TC was added to 800 μL of AgNPs.Separately, 62.5 μL of 1 mM TMAT or TMA, respectively, was added to 325μL of deionized water and stirred. The linker solution was then added tothe stirring Au/TC solution and allowed to stir overnight.

Lipid Preparation

Lipids were prepared as per the protocol reported by Ip et el. (Ip, S.;Maclaughlin, C. M.; Gunari, N.; Walker, G. C. Langmuir 2011, 27,7024-7033) Briefly, in a 3:1 chloroform/methanol solution, DOPC, ESM andChol were mixed in a 2:2:1 molar ratio (DEC221), respectively, to afinal mass of 10.7 mg. One milligram aliquots were placed in glass vialsand dried under a stream of argon gas until the solvent evaporated, anda film of lipid was visible on the bottom of each vial. The vials wereleft to dry under vacuum overnight to remove any remaining solvent, thenbackfilled with argon and capped. DEC221 lipids were stored at −20° C.until use.

The encapsulation of nanoparticles by lipids was achieved by sonicatingnanoparticles in a suspension of multilamellar vesicles (MLV) of DEC221for 45-60 min at 50° C.,

Lipid Encapsulation of J-aggregate/Nanoparticle Complex

Prior to encapsulation, DEC221 was thawed and hydrated with water to aconcentration of 1 mg/mL. The lipids were then warmed in a 50° C. waterbath. The lipids were agitated with vortex mixing every 10 min for 30min until a multilammelar vesicle suspension was formed. Thefunctionalized nanoparticles were then encapsulated by adding 1 mL ofparticles to 1 mL of the DEC221, and sonicating at 50° C. for 60 min oruntil clear. Sonication of the MLV under these conditions in the absenceof particles has been shown to produce unilamellar vesicles (ULV) <100nm in diameter (see Lapinski, M. M.; Castro-Forero, A.; Greiner, A. J.;Ofoli, R. Y.; Blanchard, G. J. Langmuir 2007, 23, 11677-11683. AndMaulucci, G.; De Spirito, M.; Arcovito, G.; Bolfi, F.; Castellano, A.C.; Briganti, G. Biophys. J. 2005, 88, 3545-3550).This transformationcan be observed visually; the MLV suspension appears cloudy, while theULV suspension appears almost clear, since the size of the vesicles inthe ULV suspension has become smaller than the diffraction limit ofvisible light, and consequently scatters significantly less light thanthe MLV suspension. Here, it is demonstrated that sonication of the MLVsuspension in the presence of metal nanoparticles results innanoparticles being encapsulated by a lipid bilayer.

All dye-lipid-particle products underwent two cleaning steps before anymeasurements were made. Each cleaning step involved centrifugation ofthe particle/vesicle suspension at 4500 RPM for 5 minutes using adesktop microcentrifuge. This settled the particles to the bottom of themicrocentrifuge tubes, and the supernatant was removed and retained in aseparate container. The particles were then resuspended to the sameconcentration in 18 MΩ-cm water.

Instrumentation and Measurement

UV-Vis Spectroscopy was performed on a Varian Cary 5000 UV-Vis-NIRspectrophotometer. Particles were placed in a 1-cm-path-length blackwail cuvette and spectra were collected at a scan speed of 240 nm/susing 18.2 MΩ·cm water as a blank. Spectra were used to confirm theplasmon shift and J-aggregate dip or peak on silver nanoparticles,respectively.

Transmission Electron Microscopy (TEM) was performed on a Hitachi H-7000TEM instrument operating at 100 kV. Samples were prepared by placing adroplet of aqueous solution containing the particles on the grid. Asmall droplet of 2% uranyl acetate solution was added to the larger oneon the grid, which was then allowed to air dry.

Raman measurements were carried out on a Renishaw InVia Confocal RamanSpectrometer, equipped with a research grade Leica microscope and 50×long-range objective. Coherent 407 nm and 514 nm lasers were used.

Data was collected with WiRe 2.0 and analyzed with GRAMS Suite software.

Theoretical Methods

The optical response of the hybrid nanostructure was investigated usingfinite difference time domain calculations using FDTD solutions(Lumerical Inc). The dielectric function of the TC dye was obtained viaextinction cross section of the TC dye in the J-aggregate state. Thiswas easily accomplished by titrating a divalent cationic salt into a 10μm TC aqueous solution, which gave rise to almost complete J-aggregateformation once the salt concentration reached 1 mM.

For accurate modeling of the thin spacer and J-aggregate layer, a meshsize of 0.3 nm is used. The simulation domain is terminated by perfectlymatched layer (PML) for minimal reflections. To calculate the absorptionand scattering cross sections of the hybrid structure, we employed theformalism of the total field scattered field (TFSF).

Results and Discussion Theoretical Results

In the FDTD calculations, the silver core is modeled by a fit to theexperimental data of Palik (Palik, E. D. Handbook of Optical Constantsof Solids; Academic press, 1998; Vol. 3) whereas the dielectric constantof the J-aggregate, ϵ_(jagg) is modeled using a Lorentz line shape givenas follows

$\begin{matrix}{ɛ_{jagg} = {ɛ_{\infty} + \frac{f\; \omega_{0}^{2}}{\omega_{0}^{2} - \omega^{2} - {i\; {\omega\gamma}_{jagg}}}}} & (1)\end{matrix}$

where f is the reduced oscillator strength, ω₀ represents the resonantangular frequency and γ_(jagg) is the line width. The permittivity ofthe modeled J-aggregate layer producing the best fit to the experimentalextinction data is shown in FIG. 1 a, The following parameters resultedin the best fit to the experimental data: f=0.33, ω₀=4.023×10¹⁵ , γ_(jagg)2.4×10¹⁴ and ϵ_(∝)=1.769. The anisotropy of the J-aggregateresponse is neglected in this analysis; while this is not rigorous, thebehavior is dominated by the radial permittivity component in thequasi-static regime (see supporting information). This is close to thepresent regime of operation and so we expect little deviation from thesecalculations for a full anisotropic model.

In this disclosure the inventors exploit the epsilon near zero (ENZ)phenomenon. It is noted that the real part of ϵ_(jagg) approaches zeroat a wavelength of 435 nm. This effect can result in strong local fieldenhancement at the interface between two different materials, asdictated by the boundary condition ϵ₁E₁=ϵ₂E₂, where ϵ_(i) and E_(i)(i=1,2) are the permittivity and normal component of the electric fieldat the interface. Thus as the permittivity of one medium approacheszero, the field strength in that medium is expected to increasesignificantly, However, it should be noted that the maximum enhancementachievable is limited by the non-zero imaginary part of ϵ_(jagg).

Given that the SERS scattering intensity varies according to theelectric field around the particle, the wavelength at which to irradiatethe particle was chosen carefully to optimize the ratio between thedielectric constants of the metal and dye. The calculations optimizedfor the maximum field intensity, and the results are shown in FIG. 1 a.The maximum field intensity for the J-aggregate around the silverparticle is 420 nm, and is sharply resonant around that wavelength.

In practice, the peak is too narrow to accommodate all Stokes-shiftedpeaks for the TC dye. We are interested mostly in enhancement of thepump electromagnetic radiation with regards to the plasmonic particle.We therefore chose to irradiate the particle at a wavelength of 407 nm,which falls very close to the plasmon resonance for this particle size,which lies around 410 nm. The brighter Stokes-shifted peaks then fallvery close to the field intensity peak. This wavelength matchingapproach is what gives rise to the large enhancement factors in terms ofelectromagnetic field around the particle.

To summarize, the following steps are followed to determine the optimumexcitation wavelength to irradiate nanoparticles to give the enhancedSERS signals:

The dielectric constant, ϵ, is calculated from the absorption crosssection for the dye in its aggregated state, (here we used UV-Vis),calculation as shown in top of FIG. 1 a, absorption data shown in FIG. 4(top, red curve). This is done by inducing some free, monomeric dye intoaggregates (ordered structures) by adding some salt. No particles are inthese spectra. It is assume the self-assembled structure is the same asthe one that forms on the particle, and therefore use that as the modeldielectric for the calculations/simulations).

It is then observed where the real portion of the dielectric constant isclose to zero. The imaginary portion of the dielectric constant isresponsible for unwanted absorption, and therefore should also beminimized. The wavelength that optimizes these effects is identified asthe target wavelength, as shown in top of FIG. 1a to be ˜425 nm for theexample above

Generate a particle that has a plasmon resonance near this wavelength,this is done by tuning geometry or composition of the particle

The dielectric constant of the metal is then obtained from literaturedata (see Ralik reference above)

The field intensity is calculated for the dye-linker-metal composite viaFDTD simulation for all wavelengths making use of the boundary conditionϵ₁E₁=ϵ₂E₂, to generate a plot similar to the bottom one in FIG. 1a

Optimal wavelength is chosen to be one with largest E-field, this is thetarget irradiation wavelength, which should fall close to the plasmonresonance of the particle

Lastly, choose a practical laser (not all wavelengths are possible,obviously) to excite the composite nanoparticles in solution. Prefer toexcite the particle as close as possible to this peak in top of FIG. 1a, while ensuring stokes shifted light from Raman scattering still fallswithin that peak and is enhanced as well.

FIG. 2 shows how the interaction distance between the particle and theJ-aggregate dye layer affects the field intensity in the particle anddye layer. As expected, the field drop-off is approximately exponentialas the layer is further removed from the particle surface. However, wefound that the relationship of field strength with respect to distancefrom the particle is heavily dependent on the wavelength. FIG. 3demonstrates that at the resonant wavelength, the field intensity isgreatly attenuated with respect to distance from the surface. However,at the non-resonant wavelength, the difference is negligible across thedistances considered, as the dielectric constant of the J-aggregate dyebecomes relatively large at wavelengths red of ˜450 nm.

This generates significant field confinement within the Li-aggregate dyewhen irradiated at 407 nm, giving values of |E| of over 800—almost twicethe field generated by the surface of the particle with no dye layerpresent. FIG. 3 shows, however, that there is negligible fieldenhancement at the non-resonant wavelength of 514 nm, where thedielectric of the dye is comparable to that of the metal. It is thestrong confinement within the dye layer as a result of the ENZphenomenon that gives rise to the large increase in SERS scatteringintensity as shown in the following section.

Experimental Results

The inventors prepared a particle which is able to exploit this unusualoptical behaviour and generate a strong Raman signature from theJ-aggregate dye monolayer that surrounds it. We first synthesized silvernanoparticles which were approximately 40+/−5 nm in diameter, confirmedby TEM. These were then functionalized with the J-aggregate layer. Giventhe lack of chemistry available to bind this particular dye to thesurface of the particle, we employed a cationic linker similar toKometani et al, (Yoshida, A.; Yonezawa, Y.; Kometani, N. Langmuir 2009,25, 6683-9) which directed the TC dye to self-assemble on the surface ofthe particle in the J-aggregate state. This strategy is applicable toany anionic J-aggregate forming dye. In order to improve particlestability, the functionalized particles were then encapsulated within alipid bilayer. An outline of the synthetic route to generate theseparticles is shown in Scheme 1. The plasmon peaks and generation of theJ-aggregates were confirmed via UV-Vis, as shown in FIG. 4. Twodifferent lengths of linker were used in order to validate therelationship between field enhancement and distance of the J-aggregatefrom the silver particle surface.

The shortest linker, TMAT, was not commercially available, and wasinstead synthesized by acid hydrolysis of an acetylthiocholineprecursor. The multi-layer particles were functionalized by the dye andspacer layer without particle aggregation. In addition to thecharacteristic ‘peak’ of the J-aggregate in the UV-Vis spectra, itspresence was confirmed by ξ-potential measurements and visuallyidentified via HRTEM. J-aggregates did not form on as-synthesizedcitrate-capped particles that were not functionalized with a cationiclinker.

The particles functionalized with the TMA linker had a somewhat sharperJ-aggregate peak in the UV-Vis spectra compared to the TMATfunctionalized particles. Given the more favourable enthalpicinteractions between the aliphatic portions of the longer TMA moleculesin comparison to the very short TMAT molecule, we postulate that therewas greater SAM density when using the longer linker. Given a discretecharge per linker molecule, the TMA functionalized AgNPs were betterable to accommodate J-aggregate formation of the anionic TC dye, whichin turn generates a larger peak in the UV-Vis spectrum for theseparticles.

The SERS spectra of the two types of particles are shown in FIG. 5. TChas been previously shown to give a rather weak Raman signature attraditional pump wavelengths (Kitahama, Y.; Tanaka, Y.; Itch, T.;Ishikawa, M.; Ozaki, Y. Bull. Chem. Soc. Jpn. 2009, 82, 1126-1132) withsome improvement as the dye excitation grows close to the J-aggregateresonance near the 458 nm wavelength. The strong peaks in the 400-1000cm⁻¹ region are indicative of J-aggregate formation within the proximityof the particle. This is due to enhancement of out of plane vibrationalmodes of the Albrecht A term through the resonance Raman effect. FIG. 5demonstrates SERRS signal at the resonant wavelength of 407 nm is overan order of magnitude greater in intensity as compared to the particlesirradiated at the off resonant wavelength, and considerably brighterthan when irradiated at wavelengths of 458 and 488 nm.

The SERS intensity difference between different separation distancesfrom the surface of the particle also closely followed simulations. TheTMAT and TMA molecules were approximated to be 0.5 and 2 nm in length,respectively, and the J-aggregate monolayer thickness is approximately 1nm. The relationship between Raman scattering intensity, I, and fieldintensity, /E/, is often approximated to be I∝//E/⁴. The ratio of thefield strengths at the two in distances as calculated by the FDTDsimulations is 1.15. We can calculate the experimental value for theratio of the field intensity using the following equation,

(I _(TMAT) /I _(TMA))=(|E|_(TMAT) /|E| _(TMA))⁴.   (2)

Using the average ratio of the peak height between the two thicknesseswas taken over eight major peaks across the spectrum, the ratio of thefield strengths was found to be approximately 1.14, which agrees wellwith the values generated by theory. However, at 514 nm, we see anegligible difference in terms of optical brightness between the twospacer layers, once again in accordance with the FDTD simulationsperformed.

It is interesting that while there may have been a greater amount of dyeon the surface of particles functionalized with the TMA in comparison tothe TMAT, the signal generated seems to be much more strongly related tothe distance between the dye layer and the particle, as seen in thesignificantly brighter SERS spectrum for the TMAT functionalizedparticles at 407 nm.

Encapsulation with Stabilizing Coating

The encapsulation of these particles was performed with a lipid mixtureof sphingolipid and cholesterol, which produced robust particles thatproved mechanically stable over the course of several months. TEM imagesof the particles with and without the lipid bilayer around the particleare shown in FIG. 6. The particles settled somewhat over the course ofseveral weeks, but were easily resuspended with simple shaking of thevial they were contained in.

The vesicle confers several advantages to this platform over ‘naked’particles. Firstly, the particles can be washed and excess dye can beremoved from solution, something that is impossible to do without thepresence of the vesicle to prevent rupture of the J-aggregate monolayer.This allows facile removal of excess dye from solution, reducingtoxicity in biological systems. Likewise, the presence of the bilayeralso ‘traps’ the J-aggregate dye within it, preventing interaction withother species which may induce undesired chemical or biological effects,while preventing degradation of optical properties due to breakup of themulti-shell nanoparticle structure.

The lipid vesicle surrounding the particle is an ideal platform forfurther modification, be it other Raman dyes or targeting moieties.These can easily be accommodated within the vesicle either by covalentattachment to the lipid anchor or physical incorporation into thealiphatic portion of the bilayer.

The lipid encapsulation of these J-aggregate functionalized particlesentraps the dye monolayer within the vesicle, and prevents dye leakagefrom the particle, as shown in FIG. 7. This allows for limitedinteraction of the dye with other species in solution, andsimultaneously promotes mechanical stability of the complex. These modelSERS particles serve as a platform to generate solution-stablebiosensors for cell surface marker detection and other applications

The above non-limiting exemplary example used silver nanoparticles, thedye 3,3′-disulfopropyl-5,5′-dichloro-9-ethyl-thiacarbocyanine sodiumsalt (TC) which forms the J-aggregate structure on the silvernanoparticles, the linkers thiocholine (TMAT) and TMA and a stabilizingcoating layer prepared from DOPC, ESM and Chol were mixed in a 2:2:1molar ratio, it will be appreciated by those skilled in the art thatthese are only examples and many alternatives of each may be used asdiscussed below.

Noble Metal Nanoparticles

Nanoparticles of the invention can be composed of materials other thangold nanoparticles. The nanoparticles are noble metal nanoparticles andmay be gold, silver, copper, nickel, palladium, platinum, ruthenium,rhodium, osmium, iridium, or an alloy of any of the foregoing metals. Invivo applications require that the SERS moiety to be biocompatible, andin such applications, the preferred metal would likely be silver orgold, but might also be an alloy of gold and silver, or gold andplatinum, or core-shell structures of two metals where the shell metalis gold. It is possible that a silver particle with a gold coating wouldbe suitable.

In terms of size, nanoparticles suitable for the invention are somewherebetween 2 nm to about 1000 nm, but more likely greater than 5 nm andless than 900 nm. Preferably, nanoparticles are between about 5 nm andabout 300 nm, or between about 5 nm and about 100 nm. It thought thatthe most likely to be preferred diameter is between about 20 nm andabout 100 nm. A very preferred range is from 20 nm to about 100 nm.While spherical nanoparticles are preferred, other shapes may be usedsuch as elliptically-, or rod-shaped nanoparticles.

Linkers and Linker Lengths

While the linker in the above example is a separate molecule from thedye molecule, it will be appreciated that under certain circumstancesthe linker may be part of dye molecule itself rather than a separateentity, for example, if the dye contains chemistry such as a thiol groupthat would allow it to adsorb directly to the particle, but if presentcan range from 0.1nm to 10 nm in length. The type of linker could beanionic, cationic or neutral depending on the chemistry of the aggregateforming dye that is used. Potential suitable linker agents includethiocholine, trimethylammonium conjugated alkanethiols, acrylates,NN-trimethyl(alkyl)ammonium, tetrabutylammonium, tetratmethylammoniumbrominde, cetyltrimethylammonium bromide, citrates, poly methacrylate,ascorbic acid, DNAs, 2-mercaptopropionic acid, 16-mercaptohexadecanoicacid, dodecyl sulfate, amino acides, homocystine, cysteine, andglutathione.

It should also be noted that the dye used in the present system could beincorporated into the lipid bilayer in several ways, for example, butnot limited to, through physical linkage to a lipid-molecule, or throughphysical incorporation into the hydrophobic fatty region between lipidmolecules (in a similar manner as cholesterol). It is also feasible togenerate a J-aggregate forming dye that generates a multi-layerstructure, such as a bilayer that forms around the particle in a mannersimilar to a lipid bilayer. The dye in this case would exhibitstructural similarity to a lipid molecule, in that it would havewell-defined hydrophobic and hydrophilic regions that facilitateself-assembly into a vesicle system.

Dyes

Dyes used in the invention can be composed of any type of polymethinedye, where the polymethine dye may be, but not limited to, a cyaninedye, including but not limited to merocyanines, indocyanines,anthrocyanines, phycocyanine, isocyanines, pseudoisocyanines andthiacyanines; squaraines; perylene bisimides; thiolatedaggregate-forming dyes that adsorb directly on the surface of thenanoparticle (and hence include a linking member as part of the dyestructure); any of the above in combination, eg. A squaraine and acyanine conjugated together. Particularly useful dyes include any classof cyanine dye that forms J-aggregates or H-aggregates, or otheraggregates in which the collective absorption of the aggregate isdifferent than that of the monomeric species of that dye.

Stabilization Coating Layer

Stabilization coatings of the invention can be composed of organicself-assembled lipid vesicles. These lipid vesicles can consist ofsterols, glycerides, sphingolipids and phospholipids, as well ascombinations thereof.

Phospholipids are a class of compounds known to those skilled in theart. One type of phospholipid is glycerol-based and includes a headgroupwhich can be, for example, phosphtidyl glycerol (PG), phosphotidylcholine (PC), phosphatidic acid (PA), phophatidyl ethanolamine (PE),phosphatidyl serine (PS), phosphatidyl inositol (PI), andderivitized/functionalized forms of these such as rhodamine-PE orPEG-PE. The tail groups can be comprised of hydrogen, or an acyl chainsuch as palmitoyl, myristyol, oleoyl, stearoyl etc. The tail group canconsist of two identical groups or two different groups. For exampleDOPC has two oleoyl acyl chains and a phosphochoine headgroup joined bya glycerol backbone. DOPG has the same acyl chains but aphosphatidylglycerol headgroup. POPC has one palmitoyl and one oleoylacyl chains and a phosphatidylcholine head group.

Also used in examples described herein was sphingomyelin, asphingophospholipid. Sphingolipids include sphingosine and itsderivatives, including sphingomyelin.

Phospholipids, which include sphingophosoplipids, are known to occurnaturally. Included within this definition are other non-naturallyoccurring phospholipids that are structurally related to certain of thenaturally occurring compounds in that they are amphipathic and can formlayers for encapsulation of metal nanoparticles. Within the definitionof phospholipids are those containing phosphtidyl glycerol (PG),phosphotidyl choline (PC), phosphatidic acid (PA), phophatidylethanolamine (PE), phosphatidyl serine (PS), and phosphatidyl inositol(PI) in their headgroup and joined via a glycerol moiety to one or twofatty acyl chains. Each chain can have from 6 up to 24 carbons, and maybe branched or unbranched, although they are typically unbranched. Eachchain can include one or more double bonds or one or more triple bonds.Chains having 12 to 24 carbon atoms are more preferred.

In a preferred embodiment, the phospholipid component of a SERS complexof the invention is a bilayer.

Examples of particular phospholipids of the invention includedioleoylphosphatiylcholine (DOPC), dipalmitoylphosphatidylchoilne(DPPC), dipalmitoyl phosphatidyl glycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), distearoyl phosphatidyl ethanolamine (DSPE),dimyristoylphosphatidyl choline (DMPC), diacyl phosphatidyl glycerols,such as dimyristoyl phosphatidyl glycerol (DMPG), dipalmitoylphosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol(DSPG), diacyl phosphatidyl cholines, such as dimyristoylphosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), anddistearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, suchas dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid(DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidylethanolamines such as dimyristoyl phosphatidyl ethanolamine (DMPE),dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidylethanolamine (DSPE),distearoylphosphatidylethanolamine-polyethyleneglycol (DSPE-PEG),dipalmitoylphosphatidylethanolamine-polyethyleneglycol (DPPE-PEG),dioleoylphosphatidylethanolarnine-polyethyleneglycol (DOPE-PEG), whereinthe PEG chain can range from 200 Da to 10000 Da molecular weight, butmore likely greater than 350 Da and less than 5000 Da. Preferably, PEGchain lengths would be in the range of 1 kDa to 5kDa molecular weight.Additionally, while one end of the PEG chain is joined to the lipidspecies, the other end of the PEG chain may be functionalized by othergroups such as a methyl group, carboxylic acid group,N-hydroxysuccinimide group, maleimide groups, cyanur group, etc, toimpart chemical functionality to the outer surface of the encapsulatedparticle, or to facilitate further chemical modification of the PEGchain, for instance to attach a targeting species.

Sphingolipids are lipid molecules with a backbone consisting of asphingoid base (itself containing an acyl chain) to which is bound afatty acyl chain and a headgroup. The fatty acyl chain can contain 6-24carbons, and may be branched or unbranched. This chain can contain 1 ormore double bonds and/or one or more triple bonds and be cis or transisomers. Chains of 12 to 24 carbons are preferred. Headgroups can, forexample, be hydrogen yielding ceramides, or phosphocholine yieldingsphingomyelin. A preferred embodiment of the invention includessphingomyelin.

Stabilization coatings of the invention can consist of inorganic oxides.Such inorganic oxides may include semiconductor oxides and oxides oftransition metals. Two examples of such oxides are silicon or titanium.These oxides would be nucleated and deposited on the surface of thedye-coated nanoparticles in a shell with a thickness of 1-100 nm butmore likely in the range of 5-20 nm. While not being bound by any theoryor speculation, it is believed that confining the dye aggregate close tothe metal nanoparticle keeps the dye molecules communicating with eachother in the aggregate and the metal nanoparticle. By confining andmaintaining the aggregate in contact with the metal nanoparticleadvantageously results in suppression of fluorescence.

Ligands Bound to Stabilization Coating Layer

A SERS complex of the invention can include an additional ligand, onethat has the ability to recognize and bind to partner of the ligand. ASERS complex having such a ligand can thus bind to the ligand partner.An example of such a ligand, one illustrated by results describedherein, is an antibody (SERS complex ligand) which binds to an antigen(target).

In the results illustrated by laboratory examples carried out anddescribed herein, the ligand was incorporated into an already preparedSERS complex. In one example, the SERS complex was prepared with aphosholipid containing a functional group to which the ligand could becovalently linked. The ligand was then linked to the functional group ofthe phospholipid in the encapsulating layer of the SERS complex. Inanother example, the ligand was linked to a phosholipid that wassubsequently incorporated into the encapsulating layer of an existingSERS complex. Yet another example uses a ligand that is attached to asilica shell which encapsulates the existing SERS complex. Each approachsuccessfully produced SERS complexes having ligands that were able torecognize their targets. The skilled person will recognize here that ananchor covalently linked to the ligand is not necessarily a phospholipidbut that it has to be e.g., a molecule compatible with the encapsulatinglayer of the SERS complex so that it can stably incorporated into orconjugated onto said layer.

Various types of moieties that can act as a ligand when covalentlylinked to a component of the encapsulating layer of the SERS complex area nucleotide, a nucleic acid molecule, a DNA molecule, an RNA molecule,an aptamer, a peptide, a protein, an amino acid, a lipid, acarbohydrate, a drug, a drug precursor, a drug candidate molecule, adrug metabolite, a vitamin, a synthetic polymer, a receptor ligand, ametabolite, an immunoglobulin, a fragment of an immunoglobulin, a domainantibody, a monoclonal antibody, a VH domain, a VL domain, a singlechain antibody, a nanobody, a unibody, a monobody, an aftbody, a DARPin,an anticalin, a ¹⁰Fn3 domain, a versabody, a Fab fragment, a Fab′fragment, an Fd fragment, an Fv fragment, an F(ab′)₂ fragment, and an Fcfragment, a proteinaceous binding molecule having an antibody-likefunction, a glubody, a protein based on the ankyrin scaffold or thecrystalline scaffold, an AdVectin, a tetranectin, an avimer, a peptoid,or a cell surface marker.

A ligand can alternatively be defined in terms of the moiety or targetwith which it binds or which it selectively captures. A ligand can thusbe one that selectively binds to a cell, a virus, bacteria, a spore, atoxin, a protein, a peptide, and amino acid, an antigen, a lipid, anucleic acid, a polynucleotide, an oligonucleotide, a drug, or anexplosive.

The skilled person will appreciate that e.g., an antibody can be linkedto the SERS complex so as to target an antigen, in one context, while inanother context, an antigen might be linked to the SERS complex as aligand which targets an antibody.

Phospholipids of the encapsulating layer of a SERS complex of theinvention are amphipathic. Hydrophilic portions of phosholipids aresituated towards the exterior of the layer and a ligand is linked sothat it can effectively recognize its partner. In one of the examplesdescribed herein, COOH-PEG-DSPE (DSPE=distearoylphosphatidylethanolamine) was incorporated into the encapsulatingphospholipid layer during production of the SERS complex. The carboxylgroup was then activated and covalently coupled to an antibody in aconventional manner. In another example, a F(ab′)₂ fragment wasgenerated and covalently linked to maleimide-PEG-DSPE through the thiolgroup of the cysteine residue of the fragment. The F(ab)-PEG-DSPE wasthen incubated with a SERS complex to be incorporated into itsencapsulating layer. The skilled person thus appreciates that the ligandis linked in these cases to the outwardly located lipid headgroup andoriented outwardly of the phospholipid layer of the SERS complex topermit binding with the target of the ligand.

Encapsulation of Nanoparticle Composites by Lipid Bilayers

Given the foregoing results, the skilled person would be capable ofpractising the invention in its various aspects, including variations asdescribed below.

The foregoing description of the preferred embodiments of the inventionhas been presented to illustrate the principles of the invention and notto limit the invention to the particular embodiment illustrated. It isintended that the scope of the invention be defined by all of theembodiments encompassed within the following claims and theirequivalents.

1. A method of producing exitonic semiconductor noble metal compositenanoparticles for enhanced Raman scattering, comprising: preparing asolution of dye molecules and linker molecules; adding the solution ofdye and linker molecules to a solution of noble metal nanoparticlesunder conditions selected to induce self-assembly of the linker and dyemolecules to produce composite particles having a layer of the linkermolecules attached to a surface of the noble metal nanoparticle and alayer of the dye molecules attached to the layer of linker molecules,the dye molecules being selected such that they interact cooperativelywith each other and with the surface, when bound to the linker layer, toproduce an optical absorption spectrum that is shifted in wavelengthrelative to an absorption spectrum of a single dye molecule;encapsulating the composite particles in a stabilizing andfluorescence-reducing coating layer; and washing the encapsulatedcomposite particles.
 2. The method according to claim 1 wherein saidnoble metal nanoparticles are any of gold, silver, copper, nickel,palladium, platinum, ruthenium, rhodium, osmium, iridium, or an alloy ofany of the foregoing metals.
 3. The method according to claim 1 whereinsaid linker molecules have a length in a range from about 0.1 nm toabout 10 nm.
 4. The method according to claim 1 wherein said linkermolecules are separate molecules from said dye molecule and include anyone of one or combination of thiocholine, trimethylammonium conjugatedalkanethiols, acrylates, NN-trimethyl(alkyl)ammonium,tetrabutylammonium, tetratmethylammonium brominde,cetyltrimethylammonium bromide, citrates, poly methacrylate, ascorbicacid, DNAs, 2-mercaptopropionic acid, 16-mercaptohexadecanoic acid,dodecyl sulfate, amino acids, homocysteine, homocystine, cysteine,cysteine, cysteine, and glutathione and derivatives thereof.
 5. Themethod according to claim 1 wherein said linker molecules are amolecular portion of the dye molecule, said molecular portion includinga moiety which binds to the surface of the noble metal nanoparticle. 6.The method according to claim 5 wherein said dye includes thiolatedaggregate-forming dyes containing said molecular agent that adsorbdirectly on the surface of the nanoparticle.
 7. The method according toclaim 1 wherein said dye includes any one of polymethine dye, where thepolymethine dye may be, but not limited to, a cyanine dye, including butnot limited to merocyanines, indocyanines, anthrocyanines, phycocyanine,isocyanines, pseudoisocyanines and thiacyanines; squaraines; perylenebisimides; any of the above in combination, e. g., a squaraine and acyanine conjugated together.
 8. The method according to claim 1 whereinsaid dye includes all classes of cyanine dye that form J-aggregates,H-aggregates, aggregates in which a collective absorption of theaggregate is different than that of the monomeric species of that dye.9. The method according to claim 1, wherein said stabilizing coating isa lipid bilayer, said lipid bilayer being any one of phospholipid alone,sphingolipid alone, phospholipid and sphingolipid, phospholipid andsterol, sphingolipid and sterol, phospholipid and sphingolipid andsterol, wherein said phospholipid is one type or a mixture ofphospholipids, said sphingolipid is one type or a mixture ofsphingolipids, and said sterol is one type or a mixture of sterols, toform a mixture.
 10. The method according to claim 9 wherein saidphospholipids have one or more hydrocarbon chain(s) being any one orcombination of saturated, monounsaturated, and polyunsaturated.
 11. Themethod according to claim 9 wherein said phospholipids and sphingolipidshave headgroups selected from the group consisting of phosphatidylcholine (PC), phosphatidyl ethanolamine (PA), phosphatidyl inositol(PI), and phosphatidyl glycerol (PG).
 12. The method according to claim9 wherein said phospholipids, sphingolipids and sterols have headgroupscan be positively charged, negatively charged, zwitterionic, or neutral.13. The method according to claim 9 wherein said phospholipids,sphingolipids and sterols include chemically modified tail groups and/orchemically modified headgroups.
 14. The method according to claim 9wherein said phospholipids are any one or combination of naturallyoccurring or synthetic, and wherein said sphingolipids are any one orcombination of naturally occurring or synthetic, and wherein saidsterols are any one or combination of naturally occurring or synthetic.15. The method according to claim 9 wherein the encapsulating stepcomprises adding detergent molecules in a suspension of lipids intowhich the nanoparticles are immersed to form the lipid bilayer aroundsaid nanoparticles.
 16. The method according to claim 9 includingcontrolling a distribution of charged lipids in said lipid bilayer byaddition of acids and/or salts in a suspension of lipids into which thenanoparticles are immersed to form the lipid bilayer around saidnanoparticles.
 17. The method according to claim 9 wherein lipidencapsulated dye aggregate coated noble metal nanoparticles areseparated from unbound phospholipids and organic dye molecules bycentrifugation.
 18. The method according to claim 9 wherein said dyeaggregate coated noble metal nanoparticles are capped by positively ornegatively charged ligands with lipids.
 19. The method according toclaim 1, wherein the stabilizing coating encapsulating a nanoparticlehas a ligand covalently linked thereto wherein the ligand is anantibody, an antibody fragment, or other targeting ligands toselectively target cell surface receptors and sub-cellular markers. 20.The method according to claim 19, wherein said other targeting ligandsincludes any one or combination of antibody fragments, peptides, DNA,RNA, proteins, affibodies, monobodies, drugs, cell surface markers, andsub-cellular markers.
 21. The method according to claim 1 wherein saidencapsulated dye aggregate coated noble metal nanoparticles areconjugated to monoclonal antibodies and other targeting ligands usingone or both of physical and chemical means of associating these witheither the gold particle core, or the phospholipid encapsulating saidgold nanoparticles.
 22. The method according to claim 1, where thestabilizing coating is a lipid bilayer, where the dye is housed withinthe bilayer structure itself, on either surface of the bilayer, or theuse of an ordered structure -forming dye modified so as to be conjugatedphysically to a lipid component.
 23. The method according to claim 1wherein said composite has a dielectric constant having wavelengthdependent real and imaginary parts value which are approximately equaland approach zero at a given wavelength, and wherein excitation of saidcomposite by a laser excitation source having a wavelength near thegiven wavelength results in an enhanced SERS signal compared toexcitation of the same nanoparticle in which the dye does not form anordered structure with a shifted absorption spectrum.
 24. The methodaccording to claim 1 wherein the stabilization coating has a thicknessin a range from about 0.1 nm to about 100 nm.
 25. The method accordingto claim 1 wherein said stabilizing coating is an inorganic oxideselected from semiconductor oxide and transition metal.
 26. The methodaccording to claim 1, wherein said stabilizing coating is an oxide ofany one of silicon, titanium and mixtures thereof.
 27. A method ofproducing enhanced Raman scattering with exitonic semiconductor noblemetal composite nanoparticles produced according to the method of claim1, comprising: identifying the optimal excitation wavelengths for fieldenhancement through a theoretical simulation that includes the complexdielectric constants of the metal and the molecular aggregate, wherein awavelength near where the plasmon is observed is matched with theexcitation wavelength (or stokes shifted wavelength) and a wavelengthnear where the real and imaginary components of ϵ_(jagg) areapproximately equal and approach zero is matched with the Stokes shift(or excitation wavelength) and identifying the plasmon through acombination of theoretical simulation and experimental measurement, anddepends on the size, shape, composition and environment of the metalnanoparticle. demonstrating the field enhancement by measuring Ramanscattering strength with excitation at different wavelengths.